Single-cycle virus for the development of canine influenza vaccines

ABSTRACT

The present invention relates to compositions and methods for the treatment and prevention of canine influenza virus (CIV) and CIV-related pathology. The present invention is based in part upon the discovery that one or more mutations in segment 4 of the viral genome produces a single cycle infectious CIV (sciCIV). The sciCIV does not allow for the production of infectious progeny, but is able to induce a CIV-specific immune response.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. national phase application filed under 35 U.S.C. § 371 claiming benefit to International Patent Application No. PCT/US16/47726, filed Aug. 19, 2016, which is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/207,579, filed Aug. 20, 2015, each of which application is hereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Canine influenza virus (CIV) is a recently emerged virus that causes acute respiratory disease in dogs. CIV was first isolated in 2004 from racing greyhound dogs during a respiratory disease outbreak at a Florida racetrack. Subsequently outbreaks were reported at greyhound racetracks and among other breeds of pet dogs. The H3N8 CIV resulted from the transfer of H3N8 equine influenza virus (EIV) to dogs around 1999. These findings were surprising as dogs were thought to be refractory to infection with influenza viruses. Most dogs have no immunity to CIV and infection may therefore spread quickly in any location with concentrated dog populations. Pet dogs are the most popular companion animals living with humans, and may support the replication of multiple influenza virus subtypes and could facilitate the generation of novel virus species with pandemic potential for humans. The true risk of human infection by CIV is unknown as we do not understand the host barriers that restrict human infection.

CIV H3N2 has been previously found in dogs in China, Korea and Thailand, where it has been circulating since it emerged in late 2005. The H3N2 CIV has been recently introduced (2015) in the USA, most likely through the transport of infected rescue dogs from Korea and is now spreading widely in the mid-Western states. This raise concerns about exposure of human in this country, as well as the likely generation of natural reassortants with the H3N8 CIV.

In 2006, the American Veterinary Medical Association (AVMA) called for the urgent development of an effective vaccine against CIV. A vaccine made from inactivated virus have been developed that is administered subcutaneously as two doses to reduce the severity of the CIV disease and to reduce the incidence of CIV infection in naive dogs (Nobivac, Merck). However, to date, no LAIV for CIV infections has been developed. Thus there is a need in the art for improved vaccines for CIV. The present invention satisfies this unmet need.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an immunological composition comprising a single-cycle infectious canine influenza virus (sciCIV), wherein the sciCIV comprises one or more mutations in segment 4 of the viral genome.

In one embodiment, the one or more mutations in segment 4 results in the lack of expression of HA. In one embodiment, the one or more mutations in segment 4 comprises the deletion of at least a portion of nucleotide sequence encoding HA. In one embodiment, the one or more mutation comprises the deletion of the whole nucleotide sequence encoding HA.

In one embodiment, the sciCIV is derived from H3N8 subtype of influenza A virus. In one embodiment, the composition is used for the treatment or prevention of canine influenza in a subject.

In one aspect, the present invention provides a method for treating or preventing canine influenza in a subject. The method comprises administering to the subject an immunological composition comprising a single-cycle infectious canine influenza virus (sciCIV), wherein the sciCIV comprises one or more mutations in segment 4 of the viral genome.

In one embodiment, the one or more mutations in segment 4 results in the lack of expression of HA. In one embodiment, the one or more mutations in segment 4 comprises the deletion of at least a portion of nucleotide sequence encoding HA. In one embodiment, the one or more mutation comprises the deletion of the whole nucleotide sequence encoding HA.

In one embodiment, the sciCIV is derived from H3N8 subtype of influenza A virus.

In one embodiment, the composition is used for the treatment or prevention of canine influenza in a subject. In one embodiment, the subject does not have canine influenza, and wherein the method induces immunity against one or more of: influenza A virus subtype H3N8 and influenza A virus subtype H3N2. In one embodiment, the subject is infected with at least one or more of: influenza A virus subtype H3N8 and influenza A virus subtype H3N2; and wherein the method induces a therapeutic immune response.

In one embodiment, the immunological composition is administered intranasally, intratracheally, orally, intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. In one embodiment, the subject is a dog.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1, comprising FIG. 1A and FIG. 1B, depicts the results of experiments demonstrating the generation and characterization of CIV (A/canine/NY/dog23/2009 H3N8) HA-expressing MDCK cells. FIG. 1A) HA protein detection by indirect immunofluorescence: Parental and HA-expressing influenza A/Puerto Rico 8/34 H1N1 (PR8) and A/canine/NY/dog23/2009 H3N8 (CIV) MDCK cells were fixed and stained with a PR8 anti-HA monoclonal antibody (PY102) or with a CIV anti-HA polyclonal antibody (NR-3103) and counterstained with DAPI to visualize the cell nuclei. Representative images obtained with a ×20 objective are shown. Bars, 50 μm. FIG. 1B) HA protein detection by Western blot: Parental and HA-expressing PR8 and CIV MDCK whole cell lysates were incubated with the PR8 anti-HA monoclonal antibody PY102 or the CIV anti-HA polyclonal NR-3103. A monoclonal antibody against actin was used as a loading control. The HA0 and HA1 are indicated with white or black arrows, respectively.

FIG. 2, comprising FIG. 2A through FIG. 2C, depicts the results of example experiments demonstrating the generation and characterization of sciCIV. FIG. 2A) Schematic representation of the recombinant GFP vRNA-like segment. The GFP vRNA-like segment contains the GFP open reading frame flanked by the terminal untranslated regions (thin black lines), along with the HA packaging signals (gray boxes), which are required for efficient incorporation of the GFP vRNA-like into the virus particle. Multicycle growth analysis of sciCIV in parental and HA-expressing MDCK cells. Confluent monolayers of parental and PR8 or CIV HA-expressing MDCK cells were infected (12-well plate format, triplicates) with the sciCIV at a low multipliciry of infection, MOI (0.001). At the indicated times post-infection (24, 48 and 72 h), GFP was visualized by fluorescence microscopy using a ×20 objective (FIG. 2B). Tissue culture supernatants at the same times post-infection were collected for sciCIV titration in MDCK-HA cells. Data represent the means±SDs of the results determined in triplicate. Dotted black line, limit of detection (200 FFU/ml) (FIG. 2C).

FIG. 3 depicts the results of experiments evaluating the attenuation of sciCIV. Female 6-to-8-week-old C57BL/6 mice (n=6) were infected intranasally with or with 1×10³ Focus Forming Units (FFU) of CIV A/canine/NY/dog23/2009 H3N8 wild-type (WT) or 1×10⁵ FFU of sciCIV. To evaluate viral lung replication, mice were sacrificed at days 2 (n=3) and 4 (n=3) post-infection and lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml) using an anti-NP monoclonal antibody (HB-65). Data represent the means and SD. Dotted black lines indicate limit of detection (200 FFU/ml).

FIG. 4, comprising FIG. 4A and FIG. 4B, depicts the results of experiments evaluating the induction of humoral responses by sciCIV vaccination. Female 6-to-8-week-old C57BL/6 mice were immunized with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular) or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV that was (sciCIV-UV) or was not (sciCIV) exposed to UV light on ice for 20 min (FIG. 4A); or mock vaccinated with PBS intranasally. At 14 days post-infection, mice were bled and the sera were collected and evaluated by ELISA for IgG antibodies against total viral proteins using cell extracts of MDCK cells infected with A/canine/NY/dog23/2009 H3N8 CIV WT (FIG. 4B). Mock-infected cell extracts were used to evaluate the specificity of the antibody response. OD, optical density. Data represent the means+/−SDs of the results for 4 individual mice.

FIG. 5, depicts the results of example experiments evaluating the protection efficacy of sciCIV. Female 6-to-8-week-old C57BL/6 mice (n=6) were immunized with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular), or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV that was (sciCIV-UV) or was not (sciCIV) exposed to UV light on ice for 20 min; or mock vaccinated with PBS intranasally. Two weeks post-vaccination, mice were challenged with 1×10⁵ FFU of A/canine/NY/dog23/2009 H3N8 CIV WT. To evaluate viral lung replication, mice were sacrificed at days 2 (n=3) and 4 (n=3) post-infection with A/canine/NY/dog23/2009 H3N8 CIV WT and lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml) using an anti-NP monoclonal antibody (HB-65). Dotted black lines indicate limit of detection (200 FFU/ml). Data represent the means+/−SDs.

FIG. 6 depicts the results of experiments evaluating the induction of humoral responses by sciCIV vaccination against A/Ca/IL/41915/2015 CIV H3N2: Female 6-to-8-week-old C57BL/6 mice were immunized with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular), or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV that was (sciCIV-UV) or was not (sciCIV) exposed to UV light on ice for 20 min; or mock vaccinated with PBS intranasally. At 14 days post-infection, mice were bled and the sera were collected and evaluated by ELISA for IgG antibodies against total influenza virus protein using cell extracts of MDCK cells infected with A/Ca/IL/41915/2015 CIV H3N2. Mock-infected cell extracts were used to evaluate the specificity of the antibody response. OD, optical density. Data represent the means+/−SDs of the results for 4 individual mice.

FIG. 7 depicts the results of example experiments evaluating the protection efficacy of sciCIV against A/Ca/IL/41915/2015 CIV H3N2: Female 6-to-8-week-old C57BL/6 mice (n=3) were immunized intranasally with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular), or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV or mock vaccinated with PBS intranasally. Two weeks post-vaccination, mice were challenged with 1×10⁵ FFU of CIV H3N2 wild-type (A/Ca/IL/41915/2015). To evaluate viral lung replication, mice were sacrificed at days 3 (n=3) post-challenge and lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml) using an anti-NP monoclonal antibody (HB-65). Dotted black lines indicate limit of detection (200 FFU/ml). Data represent the means+/−SDs.

DETAILED DESCRIPTION

The present invention relates to compositions and methods for the treatment and prevention of canine influenza virus (CIV) and CIV-related pathology. The present invention is based in part upon the discovery that one or more mutations in segment 4 of the viral genome produces a single cycle infectious CIV (sciCIV). The sciCIV does not allow for the production of infectious progeny, but is able to induce a CIV-specific immune response. In certain embodiments, the sciCIV is a live-attenuated CIV (LACIV).

In certain embodiments, the present invention provides a composition for the treatment and prevention of canine influenza virus (CIV) and CIV-related pathology. In one embodiment, the composition comprises a sciCIV having one or more mutations in segment 4, which natively encodes for HA protein. For example, in one embodiment, the sciCIV comprises a deletion mutant in segment 4, which results in the lack of HA expression.

In certain embodiments, the present invention provides a method for treating or preventing CIV and CIV-related pathology, comprising administering a composition comprising a LACIV. In certain embodiments, the method comprises intranasal delivery of the LACIV.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample.

As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

As used herein, by “combination therapy” is meant that a first agent is administered in conjunction with another agent. “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to the individual. Such combinations are considered to be part of a single treatment regimen or regime.

As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

The term “immunoglobulin” or “Ig,” as used herein, is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

As used herein, the term “immune response” includes T-cell mediated and/or B-cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity, and B cell responses, e.g., antibody production. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

“Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

The term “simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).

By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A,” the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

The term “normal temperature” or “normal body temperature” as used herein refers to the temperature of a healthy subject. For example, in certain instances the “normal body temperature” in a human subject is in the range of about 36° C. to about 38° C. In certain instances, in a canine subject, “normal body temperature” is in the range of about 38° C. to about 39.5° C.

The term “elevated temperature” or “elevated body temperature” as used herein refers to a temperature in a subject that is greater than the “normal body temperature” of a subject of a given organism. In certain instances “elevated body temperature” may be indicative of a fever, infection, or other illness. In certain instances, elevated body temperature in a human subject is greater than about 37° C. In certain instances, elevated body temperature in a canine subject is greater than about 38.5° C.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention provides immunological compositions and methods useful for the inhibition, prevention and treatment of canine influenza and canine influenza related diseases and disorders. In one embodiment, the immunological composition comprises a live-attenuated virus (LAV).

In one embodiment, the present invention provides a single-cycle infectious virus of a canine influenza virus. For example, it is demonstrated herein that one or more mutations in segment 4 of the CIV genome produces a sciCIV. The sciCIV of the present invention is unable to produce infectious progeny. However, the sciCIV provides antigen-specific immune responses and protection against CIV. In one embodiment, the sciCIV provides at least the same antigen-specific immune responses and protection against CIV compared to wildtype CIV. In certain embodiments, the sciCIV provides greater antigen-specific immune responses and protection against CIV as compared to inactivated CIV.

In general, wild-type influenza viruses contain a segmented genome with 8 segments as described in Table 1 below:

TABLE 1 Segment Gene Product 1 PB2 (Polymerase (basic) protein 2) 2 PB1 (Polymerase (basic) protein 1) 3 PA (Polymerase (acidic) protein) 4 HA (Hemagglutinin) 5 NP (Nucleoprotein) 6 NA (Neuraminidase) 7 M1 (Matrix protein 1) and M2 (Matrix protein 2) 8 NS1 (non-structural protein 1) and NEP/NS2 (non-structural protein 2)

In certain embodiments, the present invention provides an immunological composition comprising segment 4, wherein segment 4 comprises one or more mutations. For example, in certain embodiments, the immunological composition comprises a sciCIV, comprising one or more mutations in segment 4. In one embodiment, the immunological composition comprises a sciCIV, comprising a deletion mutant in segment 4 resulting in the lack of HA expression.

The present invention also provides methods of preventing, inhibiting, and treating CIV and CIV-related diseases and disorders. In one embodiment, the methods of the invention induce immunity against CIV by generating an immune response directed to CIV. In one embodiment, the methods of the invention induce production of CIV-specific antibodies. In one embodiment, the methods of the invention prevent CIV-related pathology. In one embodiment, the methods of the invention comprise administering an immunological composition comprising a sciCIV, wherein the sciCIV comprises one or more mutations in segment4, to a subject in need thereof. In one embodiment, the methods comprise administering an immunological composition to a subject in need thereof, thereby inducing immunity to CIV.

Compositions

The present invention provides immunological compositions that when administered to a subject in need thereof, elicit an immune response directed against canine influenza virus (CIV). In some embodiments, the composition includes polypeptides, nucleotides, vectors, or vaccines. Further, when the compositions are administered to a subject, they elicit an immune response that serves to protect the inoculated subject against canine influenza. As exemplified herein, the composition can be obtained in large quantities for use as a vaccine.

In one embodiment, the present invention provides compositions that are useful as immunomodulatory agents, for example, in stimulating immune responses and in preventing canine influenza and canine influenza-related pathology.

In one embodiment, the composition is a mutant CIV that induces an anti-CIV immune response. In one embodiment, the mutant CIV is a sciCIV comprising one or more mutations in segment 4. For example, in one embodiment, the sciCIV comprises a deletion mutant in segment 4, such that the sciCIV does not express HA. In one embodiment, the deletion mutant of segment 4 is lacking at least a portion of the nucleotide sequence that encodes HA. In one embodiment, the deletion mutant of segment 4 is lacking the entirety of the nucleotide sequence that encodes HA. In one embodiment, segment 4 of the sciCIV comprises HA packing signals (see FIG. 2). In certain embodiments, the sciCIV is unable to produce infectious progeny, but is still able to induce an anti-CIV immune response. In certain embodiments, the sciCIV is a live-attenuated CIV (LACIV).

In one embodiment, the sciCIV is based upon the genome of Influenza A/canine/NY/dog23/2009 H3N8. Wildtype nucleic acid sequences for each segment of Influenza A/canine/NY/dog23/2009 H3N8 and wildtype amino acid sequences for the encoded proteins are summarized in Table 2 below:

TABLE 2 Wildtype sequences for Influenza A/canine/NY/dog23/2009 H3N8 Segments Gene Products Segment 1 (SEQ ID NO: 1) PB2 (SEQ ID NO: 2) Segment 2 (SEQ ID NO: 3) PB1 (SEQ ID NO: 4) Segment 3 (SEQ ID NO: 5) PA (SEQ ID NO: 6) Segment 4 (SEQ ID NO: 7) HA (SEQ ID NO: 8) Segment 5 (SEQ ID NO: 9) NP (SEQ ID NO: 10) Segment 6 (SEQ ID NO: 11) NA (SEQ ID NO: 12) Segment 7 (SEQ ID NO: 13) M1 (SEQ ID NO: 14) M2 (SEQ ID NO: 15) Segment 8 (SEQ ID NO: 16) NS1 (SEQ ID NO: 17) NEP/NS2 (SEQ ID NO: 18)

In one embodiment, the composition comprises one or more mutations in the nucleic acid sequences of segment 4, which, in wildtype CIV, encodes HA. Thus, in certain embodiments, the composition comprises a deletion mutant of segment 4, where HA is not expressed. As described herein, the mutation of segment 4 produces a sciCIV that is unable to produce infectious progeny, but is able to induce an immune response.

In one embodiment, the sciCIV comprises segment 4 having HA packaging signals. For example, in certain embodiments, the mutant segment 4 of the sciCIV comprises packaging signals at its 5′ (80 nucleotides) and 3′ (45 nucleotides) ends. In certain embodiments, the mutant segment 4 comprises 5′ and 3′ untranslated regions (UTRs). In certain embodiments, mutant segment 4 comprises a nucleotide sequence encoding a marker protein. Exemplary marker proteins include, but are not limited to GFP, eGFP, YFP, RFP, CFP, luciferase, beta-galactosidase, and the like. For example, in certain embodiments, a nucleotide sequence encoding the marker protein replaces the nucleotide sequence encoding HA.

In one embodiment, the composition comprises nucleotide sequence that is substantially homologous to a nucleotide sequence described herein. For example, in certain embodiments, the composition comprises a nucleotide sequence that is at least 50% homologous, at least 60% homologous, at least 70% homologous, at least 80% homologous, at least 90% homologous, at least 95% homologous, at least 98% homologous, at least 99% homologous, or at least 99.5% homologous to a nucleotide sequence described herein.

In certain embodiments, the composition comprises one or more mutations in the nucleic acid sequences of segment 4, while comprising wildtype nucleic acid sequences for the rest of the segmented genome. For example, in one embodiment, the sciCIV comprises one or more mutations in segment 4 and comprises wildtype segment 1, segment 2, segment 3, segment 5, segment 6, segment 7, and segment 8.

In certain embodiments, the composition comprises one or more mutations in segment 4, in combination with one or more mutations in one or more other segments of the viral genome.

For example, in one embodiment, the composition further comprises one or more mutations in segment 8. In one embodiment, the composition comprises a deletion mutant of segment 8, such that the coding region of NS1 protein is truncated or deleted, as described in PCT Patent Application PCT/US2016/47711, filed on Aug. 19, 2016, claiming priority to U.S. Provisional Patent Application No. 62/207,576, each of which applications are incorporated by reference in their entirety.

In one embodiment, the composition further comprises one or more mutations in segment 1 and/or segment 2. In one embodiment, the composition comprises a mutation in segment 1 and/or segment 2, encoding a point mutation in PB2 and/or PB1 that render the CIV temperature sensitive. An exemplary point mutations of PB2 is N265S. Exemplary point mutations of PB1 include a K391E point mutation, a E581G point mutation, and a A661T point mutation, as described in PCT Patent Application PCT/US2016/47715, filed on Aug. 19, 2016, claiming priority to U.S. Provisional Patent Application No. 62/207,571, each of which applications are incorporated by reference in their entirety.

In certain embodiments, the composition comprises a polynucleotide comprising a deletion mutation of segment 4. The polynucleotide can be RNA or DNA. In one embodiment, the composition comprises a DNA vaccine.

The nucleic acid sequences include both the DNA sequence that is transcribed into RNA and the RNA sequence that is translated into a polypeptide. According to other embodiments, the polynucleotides of the invention are inferred from the amino acid sequence of the polypeptides of the invention. As is known in the art several alternative polynucleotides are possible due to redundant codons, while retaining the biological activity of the translated polypeptides.

Further, the invention encompasses an isolated nucleic acid comprising a nucleotide sequence having substantial homology to a nucleotide sequence of an isolated nucleic acid encoding a polypeptide disclosed herein. Preferably, the nucleotide sequence of an isolated nucleic acid encoding a polypeptide of the invention is “substantially homologous,” that is, is about 60% homologous, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably, about 95% homologous, and even more preferably about 99% homologous to a nucleotide sequence of an isolated nucleic acid encoding a polypeptide of the invention.

It is to be understood explicitly that the scope of the present invention encompasses homologs, analogs, variants, fragments, derivatives and salts, including shorter and longer polypeptides and polynucleotides, as well as polypeptide and polynucleotide analogs with one or more amino acid or nucleic acid substitution, as well as amino acid or nucleic acid derivatives, non-natural amino or nucleic acids and synthetic amino or nucleic acids as are known in the art, with the stipulation that these modifications must preserve the immunologic activity of the original molecule. Specifically any active fragments of the active polypeptides as well as extensions, conjugates and mixtures are included and are disclosed herein according to the principles of the present invention.

The invention should be construed to include any and all isolated nucleic acids which are homologous to the nucleic acids described and referenced herein, provided these homologous nucleic acids encode polypeptides having the biological activity of the polypeptides disclosed herein.

The skilled artisan would understand that the nucleic acids of the invention encompass a RNA or a DNA sequence encoding a polypeptide of the invention, and any modified forms thereof, including chemical modifications of the DNA or RNA which render the nucleotide sequence more stable when it is cell free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleotide sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleotide sequences are contemplated in the present invention.

Further, any number of procedures may be used for the generation of mutant, derivative or variant forms of a protein of the invention using recombinant DNA methodology well known in the art such as, for example, that described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Procedures for the introduction of amino acid changes in a polypeptide or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in these, and other, treatises.

According to yet another embodiment, composition of the invention, comprising the nucleic acid sequences or combination of nucleic acid sequences of the present invention, is capable of generating a CIV-specific immune response. In another embodiment, the composition of the invention, comprising the nucleic acid sequences or combination of nucleic acid sequences of the present invention, is capable of generating CIV-specific antibodies. In certain embodiments, the composition is able to protect against CIV, including H3N8 CIV and H3N2 CIV.

The invention should also be construed to include any form of a polypeptide having substantial homology to the polypeptides disclosed herein. Preferably, a polypeptide which is “substantially homologous” is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably, about 95% homologous, and even more preferably about 99% homologous to amino acid sequence of the polypeptides disclosed herein.

According to yet another embodiment, composition of the invention, comprising the polypeptide or combination of polypeptides of the present invention, is capable of generating a CIV-specific immune response. In another embodiment, the composition of the invention, comprising the polypeptide or combination of polypeptides of the present invention, is capable of generating CIV-specific antibodies. In certain embodiments, the composition is able to protect against CIV, including H3N8 CIV and H3N2 CIV.

The present invention should also be construed to encompass “mutants,” “derivatives,” and “variants” of the polypeptides of the invention (or of the DNA encoding the same) which mutants, derivatives and variants are polypeptides which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or more base pairs) such that the resulting polypeptide (or DNA) is not identical to the sequences recited herein, but has the same biological property as the polypeptides disclosed herein.

Mutant Viruses

The invention relates in part to the generation, selection and identification of mutant CIV that generate a CIV-specific immune response, and the use of such viruses in vaccine and pharmaceutical formulations. In one embodiment, the mutant virus is a sciCIV. In one embodiment, the mutant virus is a LACIV.

As described herein, in certain embodiments the mutant virus comprises one or more mutations in segment 4. For example, in one embodiment, the mutant virus comprises a deletion mutant of segment 4, where the mutant virus does not express HA. In one embodiment, the mutant virus is unable to produce infectious progeny. However, the mutant virus induces CIV-specific immune responses and antibody production, and is thus able to protect against CIV and CIV-related pathology.

Any mutant virus or strain which has at least one mutation can be selected and used in accordance with the invention. In one embodiment, naturally occurring mutants or variants, or spontaneous mutants can be selected that include at least one mutation in segment 4, as described elsewhere herein. In another embodiment, mutant viruses can be generated by exposing the virus to mutagens, such as ultraviolet irradiation or chemical mutagens, or by multiple passages and/or passage in non-permissive hosts. Screening in a differential growth system can be used to select for those mutants having at least one mutation in segment 4, as described elsewhere herein. For viruses with segmented genomes, the attenuated phenotype can be transferred to another strain having a desired antigen by reassortment, (i.e., by coinfection of the attenuated virus and the desired strain, and selection for reassortants displaying both phenotypes).

In another embodiment, mutations can be engineered into an influenza virus, including, but not limited to H3N8 CIV or H3N2 CIV using “reverse genetics” approaches. In this way, natural or other mutations which confer the attenuated phenotype can be engineered into vaccine strains. For example, deletions, insertions, or substitutions of the coding region of segment 4, encoding HA, can be engineered. Deletions, substitutions or insertions in the non-coding region of segment 4 are also contemplated. To this end, mutations in the signals responsible for the transcription, replication, polyadenylation and/or packaging of segment 4 can be engineered.

In certain instances, the reverse genetics technique involves the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative strand virus RNA which are essential for the recognition by viral polymerases and for packaging signals necessary to generate a mature virion. The recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) which can be used to transfect cells. In some instances, a more efficient transfection is achieved if the viral polymerase proteins are present during transcription of the synthetic RNAs either in vitro or in vivo. The synthetic recombinant RNPs can be rescued into infectious virus particles. The foregoing techniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24, 1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in European Patent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. patent application Ser. No. 09/152,845; in International Patent Publications PCT WO97/12032 published Apr. 3, 1997; WO96/34625 published Nov. 7, 1996; in European Patent Publication EP-A780475; WO 99/02657 published Jan. 21, 1999; WO 98/53078 published Nov. 26, 1998; WO 98/02530 published Jan. 22, 1998; WO 99/15672 published Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998; WO 97/06270 published Feb. 20, 1997; and EPO 780 47SA1 published Jun. 25, 1997, each of which is incorporated by reference herein in its entirety.

Attenuated viruses generated by the reverse genetics approach can be used in the vaccine and pharmaceutical formulations described herein. Reverse genetics techniques can also be used to engineer additional mutations to other viral genes important for vaccine production—i.e., the epitopes of useful vaccine strain variants can be engineered into the attenuated virus. Alternatively, completely foreign epitopes, including antigens derived from other viral or non-viral pathogens can be engineered into the attenuated strain.

In an alternate embodiment, a combination of reverse genetics techniques and reassortant techniques can be used to engineer attenuated viruses having the desired epitopes. For example, an attenuated virus (generated by natural selection, mutagenesis or by reverse genetics techniques) and a strain carrying the desired vaccine epitope (generated by natural selection, mutagenesis or by reverse genetics techniques) can be co-infected in hosts that permit reassortment of the segmented genomes. Reassortants that display both the attenuated phenotype and the desired epitope can then be selected.

The attenuated virus of the present invention can itself be used as the active ingredient in vaccine or pharmaceutical formulations. In certain embodiments, the attenuated virus can be used as the vector or “backbone” of recombinantly produced vaccines. To this end, the “reverse genetics” technique can be used to engineer mutations or introduce foreign epitopes into the attenuated virus, which would serve as the “parental” strain. In this way, vaccines can be designed for immunization against strain variants, or in the alternative, against completely different infectious agents or disease antigens.

For example, in one embodiment, the immunological composition of the invention comprises a mutant virus, engineered to express one or more epitopes or antigens of CIV along with epitopes or antigens of another pathogen. For example, the mutant virus can be engineered to express neutralizing epitopes of other preselected strains. Alternatively, epitopes of other viruses can be built into the mutant virus. Alternatively, epitopes of non-viral infectious pathogens (e.g., parasites, bacteria, fungi) can be engineered into the virus.

In one embodiment, the mutant viruses selected for use in the invention is capable of inducing a robust anti-CIV response in the host—a feature which contributes to the generation of a strong immune response when used as a vaccine, and which has other biological consequences that make the viruses useful as pharmaceutical agents for the prevention and/or treatment of other viral infections, or other diseases.

The viruses, which induce a CIV-specific immune response in hosts, may also be used in pharmaceutical formulations for the prophylaxis or treatment of other influenza infections, or influenza-related pathology. In this regard, the tropism of the attenuated virus can be altered to target the virus to a desired target organ, tissue or cells in vivo or ex vivo. Using this approach, the CIV-specific immune response can be induced locally, at the target site, thus avoiding or minimizing the side effects of systemic treatments. To this end, the attenuated virus can be engineered to express a ligand specific for a receptor of the target organ, tissue or cells.

Vaccine

In certain aspects, the immunological composition is useful as a vaccine, where the immunological composition induces an immune response to the antigen in a cell, tissue or mammal. Preferably, the vaccine induces a protective immune response in the mammal. As used herein, an “immunological composition” may comprise, by way of examples, a virus, a mutant virus, a single-cycle infectious virus, a live-attenuated virus (LAV), an antigen (e.g., a polypeptide), a nucleic acid encoding an antigen (e.g., an antigen expression vector), or a cell expressing or presenting an antigen or cellular component. In particular embodiments the immunological composition comprises or encodes all or part of any polypeptide antigen described herein, or an immunologically functional equivalent thereof. In other embodiments, the immunological composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination. In certain embodiments, the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.

In the context of the present invention, the term “vaccine” refers to a substance that induces anti-CIV immunity or suppresses CIV upon inoculation into an animal.

The invention encompasses vaccine formulations comprising a mutant CIV. For example, in one embodiment, the mutant CIV is a sciCIV. For example, in certain embodiments, the sciCIV is unable to produce infectious progeny. In one embodiment, the vaccine comprises a sciCIV comprising one or more mutations in segment 4, and a suitable excipient. The virus used in the vaccine formulation may be selected from naturally occurring mutants or variants, mutagenized viruses or genetically engineered viruses. Mutant strains of CIV can also be generated via reassortment techniques, or by using a combination of the reverse genetics approach and reassortment techniques. Naturally occurring variants include viruses isolated from nature as well as spontaneous occurring variants generated during virus propagation. The mutant virus can itself be used as the active ingredient in the vaccine formulation. Alternatively, the attenuated virus can be used as the vector or “backbone” of recombinantly produced vaccines. To this end, recombinant techniques such as reverse genetics (or, for segmented viruses, combinations of the reverse genetics and reassortment techniques) may be used to engineer mutations or introduce foreign antigens into the attenuated virus used in the vaccine formulation. In this way, vaccines can be designed for immunization against strain variants, or in the alternative, against completely different infectious agents or disease antigens.

In one embodiment, the vaccine formulation comprises a plurality of mutant CIV. For example, in one embodiment, the vaccine formulation may comprise one or more of the sciCIV, described herein, in combination with other mutant CIV that induce an anti-CIV immune response. For example, in one embodiment, the vaccine formulation comprises a live-attenuated CIV having one or more mutations in segment 1 and/or segment 2. In one embodiment, the vaccine formulation comprises a mutant CIV comprising a deletion mutant in segment 8.

In one embodiment, the present invention comprises a method of generating a mutant CIV, comprising contacting a host cell with a polynucleotide comprising the nucleic acid sequences of segment 4, having one or more mutations, described elsewhere herein.

Propagation of the virus in culture is known to persons in the art. Briefly, the virus is grown in the media compositions in which the host cell is commonly cultured. Suitable host cells for the replication of CIV include, e.g., Vero cells, BHK cells, MDCK cells, 293 cells COS cells, and CEK cells, including 293T cells, COS7 cells. Commonly, co-cultures including two of the above cell lines, e.g., MDCK cells and either 293T or COS cells are employed at a ratio, e.g., of 1:1, to improve replication efficiency. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and CO₂ concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2). Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and/or additional nutrients, such as L-glutamine, sodium pyruvate, non-essential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

Procedures for maintaining mammalian cells in culture have been extensively reported, and are known to those of skill in the art. General protocols are provided, e.g., in Freshney (1983) Culture of Animal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul (1975) Cell and Tissue Culture, 5th ed., Livingston, Edinburgh; Adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology-Cell Culture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam. Additional details regarding tissue culture procedures of particular interest in the production of influenza virus in vitro include, e.g., Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation. In Cohen and Shafferman (eds) Novel Strategies in Design and Production of Vaccines, which is incorporated herein in its entirety. Additionally, variations in such procedures adapted to the present invention are readily determined through routine experimentation.

Cells for production of a virus can be cultured in serum-containing or serum free medium. In some case, e.g., for the preparation of purified viruses, it is desirable to grow the host cells in serum free conditions. Cells can be cultured in small scale, e.g., less than 25 ml medium, culture tubes or flasks or in large flasks with agitation, in rotator bottles, or on microcarrier beads (e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylamine beads, such as Hillex, SoloHill, Ann Arbor) in flasks, bottles or reactor cultures. Microcarrier beads are small spheres (in the range of 100-200 microns in diameter) that provide a large surface area for adherent cell growth per volume of cell culture. For example a single liter of medium can include more than 20 million microcarrier beads providing greater than 8000 square centimeters of growth surface. For commercial production of viruses, e.g., for vaccine production, it is often desirable to culture the cells in a bioreactor or fermenter. Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors (New Brunswick Scientific, Edison, N.J.); laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).

Virtually any heterologous gene sequence may be constructed into the viruses of the invention for use in vaccines. Preferably, epitopes that induce a protective immune response to any of a variety of pathogens, or antigens that bind neutralizing antibodies may be expressed by or as part of the viruses. For example, heterologous gene sequences that can be constructed into the viruses of the invention for use in vaccines include but are not limited to epitopes of human immunodeficiency virus (HIV) such as gp120; hepatitis B virus surface antigen (HBsAg); the glycoproteins of herpes virus (e.g. gD, gE); VP1 of poliovirus; antigenic determinants of non-viral pathogens such as bacteria and parasites, to name but a few. In another embodiment, all or portions of immunoglobulin genes may be expressed. For example, variable regions of anti-idiotypic immunoglobulins that mimic such epitopes may be constructed into the viruses of the invention. In yet another embodiment, tumor associated antigens may be expressed.

Either a live recombinant viral vaccine or an inactivated recombinant viral vaccine can be formulated. A live vaccine may be preferred because multiplication in the host leads to a prolonged stimulus of similar kind and magnitude to that occurring in natural infections, and therefore, confers substantial, long-lasting immunity. Production of such live recombinant virus vaccine formulations may be accomplished using conventional methods involving propagation of the virus in cell culture or in the allantois of the chick embryo followed by purification.

Many methods may be used to introduce the vaccine formulations described above, these include but are not limited to introduction intranasally, intratracheally, orally, intradermally, intramuscularly, intraperitoneally, intravenously, and subcutaneously. It may be preferable to introduce the virus vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed, or via the natural route of infection of the parental attenuated virus.

A vaccine of the present invention, comprising a mutant CIV, for example a sciCIV, could be administered once. Alternatively, a vaccine of the present invention, comprising a mutant CIV, could be administered twice or three or more times with a suitable interval between doses. Alternatively, a vaccine of the present invention, comprising a mutant CIV, could be administered as often as needed to an animal, preferably a mammal.

Methods

The invention provides a method for treating or preventing canine influenza infection or a CIV-related disease or disorder. In one embodiment, the method comprises administering an immunological composition comprising a mutant CIV. In one embodiment, the method comprises administering an immunological composition comprising a mutant CIV comprising one or more mutations in segment 4, to a subject in need thereof. In one embodiment, the mutant CIV is a sciCIV.

In certain embodiments, the mutant CIV induces an enhanced immune response as compared to an inactivated CIV. For example, in certain embodiments, the induced immune response of LACIV is 2-fold more, 3-fold more, 5-fold more, 10-fold more, 15-fold more, 20-fold more, 50-fold more, 100-fold more, 500-fold more, or 1000-fold more, than inactivated CIV. The immune response induced the mutant CIV can be measured using standard assays. For example, in certain embodiments, the immune response induced by mutant CIV is measured by detecting the amount of CIV-specific antibodies produced in the subject following administration of mutant CIV.

The therapeutic compositions of the invention may be administered prophylactically or therapeutically to subjects suffering from, or at risk of, or susceptible to, developing the disease or condition. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

In certain embodiments, the subject is a mammal. For example, the subject may include, but is not limited to, a human, primate, cow, horse, sheep, pig, dog, cat, or rodent. In one embodiment, the subject is a dog. The method may be used to treat or prevent CIV or CIV-related pathology in any breed or species of dog. In certain embodiments, the relative amount of active ingredient in a single dose, or the frequency of doses, will vary depending on the age, sex, weight, or breed of subject (e.g. dog).

The composition may be combined with an adjuvant. An adjuvant refers to a compound that enhances the immune response when administered together (or successively) with the immunological composition. Examples of suitable adjuvants include cholera toxin, salmonella toxin, alum and such, but are not limited thereto. Furthermore, a vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such. Furthermore, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants and such. The vaccine is administered systemically or locally. Vaccine administration may be performed by single administration or boosted by multiple administrations.

Administration

In one embodiment, the methods of the present invention comprise administering an immunological composition of the invention directly to a subject in need thereof. Administration of the composition can comprise, for example, intranasal, intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

Furthermore, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

Pharmaceutical Compositions

The present invention envisions treating or preventing CIV or CIV-related pathology in a mammal by the administration of a therapeutic composition of the invention to a mammal in need thereof. Administration of the composition in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the compositions of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art.

The present invention encompasses pharmaceutical compositions comprising a mutant CIV to be used as anti-viral agents or as agents against CIV-related diseases and disorders. The pharmaceutical compositions have utility as an anti-viral prophylactic and may be administered to a subject at risk of getting infected or is expected to be exposed to a virus. For example, subjects traveling to parts of the world where CIV is prevalent can be administered a pharmaceutical composition of the invention. In certain embodiments, subjects who are expected to be in contact with other subjects at risk, can be administered a pharmaceutical composition of the invention.

The mutant CIV of the invention may be engineered using the methods described herein to express proteins or peptides which would target the viruses to a particular site. In one embodiment, where the site to be targeted expresses a receptor to a growth factor, e.g., VEGF, EGF, or PDGF, the mutant CIV may be engineered to express the appropriate growth factor or portion(s) thereof. Thus, in accordance with the invention, the mutant CIV may be engineered to express any target gene product, including peptides, proteins, such as enzymes, hormones, growth factors, antigens or antibodies, which will function to target the virus to a site in need of anti-viral, antibacterial, anti-microbial or anti-cancer activity.

Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical compositions of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, in a preferred embodiment it may be desirable to introduce the pharmaceutical compositions of the invention into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

In certain embodiments, the pharmaceutical composition is a veterinary pharmaceutical composition suitable for administration to a veterinary subject, including but not limited to a canine subject. Exemplary canine subjects include dogs, wolves, foxes, coyotes, and jackals.

In certain embodiments, the veterinary pharmaceutical composition is “palatable,” meaning an oral veterinary composition that is readily accepted by canines, including dogs, without any coaxing or with some coaxing. Palatable compositions are compositions that score at least 2 using a palatability assessment method wherein dog owners score the composition from 0 to 3, wherein dogs scoring 0 do not consume the composition; dogs scoring 1 consume the composition after some time; dogs scoring 2 consume the composition with some coaxing and dogs scoring 3 consume the composition readily. A skilled person is well-versed in these palatability standards and scoring regimes. In another embodiment, the daily dose for dogs may be around 100 mg/kg. Veterinary pharmaceutical agents that may be included in the compositions of the invention are well-known in the art (see e.g. Plumb' Veterinary Drug Handbook, 5th Edition, ed. Donald C. Plumb, Blackwell Publishing, (2005) or The Merck Veterinary Manual, 9th Edition, (January 2005)).

In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger & Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, i.e., the lung, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the attenuated virus, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. These compositions can be formulated as a suppository. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The amount of the pharmaceutical composition of the invention which will be effective in the treatment or prevention of a particular disease or disorder will depend on the nature of the disease or disorder, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1

Using plasmid-based reverse genetics techniques, a single-cycle infectious canine influenza virus (sciCIV) was developed based on the A/canine/NY/dog23/2009 H3N8 influenza virus. In this sciCIV approach, the fourth viral segment, which encodes for the receptor-binding and fusion protein hemagglutinin (HA), has been removed. Thus, upon infection of normal cells, although no infectious progeny are produced, the expression of other viral proteins occurs and is immunogenic. The nucleic acid sequences of the viral segments, and the amino acid sequences of the encoded viral proteins, used in the development of the sciCIV are provided in SEQ ID NOs: 1-18)

Experiments were first conducted to generate and characterize CIV (A/canine/NY/dog23/2009 H3N8) HA-expressing MDCK cells. HA protein was first detected by indirect immunofluorescence. Parental and HA-expressing influenza A/Puerto Rico 8/34 H1N1 (PR8) and A/canine/NY/dog23/2009 H3N8 (CIV) MDCK cells were fixed and stained with a PR8 anti-HA monoclonal antibody (PY102) or with a CIV anti-HA polyclonal antibody (NR-3103) and counterstained with DAPI to visualize the cell nuclei (FIG. 1A). HA protein was also detected by Western blot. Parental and HA-expressing PR8 and CIV MDCK whole cell lysates were incubated with the PR8 anti-HA monoclonal antibody PY102 or the CIV anti-HA polyclonal NR-3103 (FIG. 1B).

A single cycle infectious CIV (sciCIV) was generated. FIG. 2A depicts a schematic of the recombinant GFP vRNA-like segment. The GFP vRNA-like segment contains the GFP open reading frame flanked by the terminal untranslated regions (thin black lines), along with the HA packaging signals (gray), which are required for efficient incorporation of the GFP vRNA-like into the virus particle. Experiments investigating the multicycle growth of sciCIV in parental and HA-expressing MDCK cells was conducted. Confluent monolayers of parental and PR8 or CIV HA-expressing MDCK cells were infected (12-well plate format, triplicates) with the sciCIV at a low MOI (0.001). GFP was visualized at 24, 48, and 72 hours post-infection by fluorescence microscopy using a ×20 objective (FIG. 2B). Tissue culture supernatants at the same times post-infection were collected for sciCIV titration in MDCK-HA cells, which demonstrates that PR8 and CIV HA-expressing cells equally support sciCIV growth (FIG. 2C).

Experiments were conducted to evaluate the attenuation of sciCIV. Female 6-to-8-week-old C57BL/6 mice (n=6) were infected intranasally with or with 1×10³ Focus Forming Units (FFU) of CIV A/canine/NY/dog23/2009 H3N8 wild-type (WT) or 1×10⁵ FFU of sciCIV. To evaluate viral lung replication, mice were sacrificed at days 2 (n=3) and 4 (n=3) post-infection and lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml) using an anti-NP monoclonal antibody (HB-65). It was observed that no detectable amount of sciCIV was present in the lungs post-infection (FIG. 3), highlighting the safety of the sciCIV approach.

Experiments were conducted to evaluate the induction of humoral responses by sciCIV vaccination. Female 6-to-8-week-old C57BL/6 mice were immunized with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular) or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV that was (sciCIV-UV) or was not (sciCIV) exposed to UV light on ice for 20 min (FIG. 4A); or mock vaccinated with PBS intranasally. At 14 days post-infection, mice were bled and the sera were collected and evaluated by ELISA for IgG antibodies against total viral proteins using cell extracts of MDCK cells infected with A/canine/NY/dog23/2009 H3N8 CIV WT. Mock-infected cell extracts were used to evaluate the specificity of the antibody response. It was observed that sciCIV was able to induce a CIV-specific immune response (FIG. 4B, Table 3).

TABLE 3 Immunogenicity of sciCIV Immunization and dose^(a) Mean (SD) serum HAI titer^(b) PBS — ≤8 (ND) WT 10³ FFU 215.3 (64) sciCIV 10⁵ FFU 8 (0) sciCIV-UV 10⁵ FFU ≤8 (ND) Nobivac 100 μl 26.9 (8) ^(a)Virus was administered intranasally to anesthetized mice (n = 4), Nobivac was administered intramuscularly, and sera were collected at 14 days postinfection. ^(b)Four HAU of the WT virus was incubated with 2-fold serial dilutions of the indicated sera. ND, not determined

Further experiments were conducted to evaluate the protection efficacy of sciCIV. Female 6-to-8-week-old C57BL/6 mice (n=6) were immunized with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular), or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV that was (sciCIV-UV) or was not (sciCIV) exposed to UV light on ice for 20 min; or mock vaccinated with PBS intranasally. Two weeks post-vaccination, mice were challenged with 1×10⁵ FFU of A/canine/NY/dog23/2009 H3N8 CIV WT. To evaluate viral lung replication, mice were sacrificed at days 2 (n=3) and 4 (n=3) post-infection with A/canine/NY/dog23/2009 H3N8 CIV WT and lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml) using an anti-NP monoclonal antibody (HB-65). It was observed that sciCIV was protective in inducing immunity against the WT challenge (FIG. 5).

This data demonstrates that sciCIV is protective against influenza homologus A/canine/NY/dog23/2009 H3N8 CIV challenge in a mouse model. Protection efficacy with sciCIV is replication-dependent, which is attributed to both humoral responses and T cells.

Further experiments were conducted to examine the ability of the H3N8 sciCIV to induce humoral responses against A/Ca/IL/41915/2015 CIV H3N2: Female 6-to-8-week-old C57BL/6 mice were immunized with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular), or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV that was (sciCIV-UV) or was not (sciCIV) exposed to UV light on ice for 20 min; or mock vaccinated with PBS intranasally. At 14 days post-infection, mice were bled and the sera were collected and evaluated by ELISA for IgG antibodies against total influenza virus protein using cell extracts of MDCK cells infected with A/Ca/IL/41915/2015 CIV H3N2. Mock-infected cell extracts were used to evaluate the specificity of the antibody response. It was observed that the H3N8 sciCIV induced humoral responses against H3N2 CIV (FIG. 6).

Further experiments were conducted to evaluate the protection efficacy of H3N8 sciCIV against A/Ca/IL/41915/2015 CIV H3N2. Female 6-to-8-week-old C57BL/6 mice (n=3) were immunized intranasally with the CIV inactivated vaccine (Nobivac; 100 ul intramuscular), or with 1×10³ FFU of A/canine/NY/dog23/2009 H3N8 CIV wild-type (WT), 1×10⁵ FFU of sciCIV or mock vaccinated with PBS intranasally. Two weeks post-vaccination, mice were challenged with 1×10⁵ FFU of CIV H3N2 wild-type (A/Ca/IL/41915/2015). To evaluate viral lung replication, mice were sacrificed at days 3 (n=3) post-challenge and lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml) using an anti-NP monoclonal antibody (HB-65). Again, it was observed that H3N8 sciCIV was able to protect against H3N2 CIV.

Thus, the present data demonstrates that sciCIV is able to protect against the newly introduced H3N2 CIV (A/Ca/IL/41915/2015). Altogether, the present studies demonstrate that the present sciCIV approach has potential as safe and broadly protective live attenuated vaccine against CIV.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. 

What is claimed is:
 1. A method for preventing canine influenza in a subject, the method comprising administering to the subject an immunological composition comprising a single-cycle infectious canine influenza virus (sciCIV) of the H3N8 subtype of influenza A virus, wherein the sciCIV comprises one or more mutations in segment 4 of the viral genome that results in the lack of expression of HA, wherein the subject does not have canine influenza, and wherein the method induces immunity against influenza A virus subtype H3N2.
 2. The method of claim 1, wherein the one or more mutation comprises the deletion of the whole nucleotide sequence encoding HA.
 3. The method of claim 1, wherein the immunological composition is administered intranasally, intratracheally, orally, intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.
 4. The method of claim 1, wherein the subject is a dog.
 5. A method for treating canine influenza in a subject, the method comprising administering to the subject an immunological composition comprising a single-cycle infectious canine influenza virus (sciCIV) of the H3N8 subtype of the influenza A virus, wherein the sciCIV comprises one or more mutations in segment 4 of the viral genome that results in the lack of expression of HA, wherein the subject is infected with influenza A virus subtype H3N2; and wherein the method induces a therapeutic immune response.
 6. The method of claim 5, wherein the one or more mutation comprises the deletion of the whole nucleotide sequence encoding HA.
 7. The method of claim 5, wherein the immunological composition is administered intranasally, intratracheally, orally, intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.
 8. The method of claim 5, wherein the subject is a dog. 